PROJECT SUMMARY Despite decades of HIV-1 vaccine research, there are still no examples of vaccines or immunogens that consistently elicit potent broadly neutralizing antibodies (bNAbs) in animal models or in human subjects. This includes recent preclinical trials of soluble Env trimers (SOSIPs) and structure-based, scaffolded Env outer domain immunogens. To overcome this critical roadblock in vaccine design, we propose a novel strategy based on the scientific premise that since most examples of successful bNAb elicitation come from natural human infection by primary HIV-1 strains, the most likely means for eliciting bNAbs in RMs is by productive infection with SHIVs bearing primary HIV-1 Envs in their native configurations. Then, by characterizing the co- evolutionary pathways of bNAbs and the cognate SHIV Envs (?immunotypes?) that elicit them through repeated rounds of B cell receptor (BCR) engagement and affinity maturation, a ?molecular guide? for rational vaccine design can be developed and tested by both ?B lineage design? and ?reverse vaccinology? approaches. This application proposes an innovative research plan ? supported by recent discoveries in our laboratory regarding novel SHIV designs ? that advances these concepts. We propose to target the development of V1V2 epitope specific bNAbs because of unique features of their ontogeny that make them attractive for vaccine development, and because of the recent identification of Env immunotypes that bind germline and intermediate ancestor V1V2 lineage BCRs, leading to affinity maturation and neutralization breadth. Building on these promising findings, we hypothesize that co-infection of rhesus macaques with antigenically-diverse SHIVs whose Envs have been preselected to bind germline and intermediate ancestor V1V2 bNAb lineage specific BCRs, and co-immunization of RMs with homologous Env SOSIPs, will enhance BCR engagement and maturation and accelerate the development of HIV-1 V1V2 targeted bNAbs. To test this hypothesis, we propose to: (i) analyze NAb potency, breadth and epitope specificity, together with molecular patterns of Env- Ab coevolution, in 8 RMs infected by a mixture of six SHIVs bearing subtype A, B and C Envs preselected to bind germline and intermediate ancestor V1V2 bNAb lineage BCRs, compared with RMs infected by individual SHIVs bearing the same Envs; (ii) analyze NAb potency, breadth and epitope specificity, together with molecular patterns of Env-Ab coevolution, in 8 RMs infected by a mixture of six SHIVs bearing the Env from CAP256SU, which binds the germline V1V2 bNAb BCR, and its evolved variants that led to bNAb maturation in the human subject CAP256, compared with animals infected by SHIV CAP256SU only; (iii) corroborate the identification of Env immunotypes in Aims 1 and 2 responsible for bNAb induction by re-engineering selected Envs into SHIVs, followed by infection of 8 RMs and analysis of NAb potency, breadth and specificity; and (iv) construct novel Env SOSIPs comprised of immunotypes identified in Aims 1-3 and use them for vaccination in 12 RMs, followed by an analysis of vaccine elicited NAb potency, breadth and specificity.